text:
|1st Samuel 24
King James Bible
Chapter 25
And Samuel died;4 and all the Israelites were gathered together,11 and lamented4 him, and buried4 him in his house at Ramah. And David arose,4 and went down4 to the wilderness of Paran.
And [there was] a man in Maon, whose possessions [were] in Carmel; and the man [was] very great, and he had three thousand sheep, and a thousand goats: and he was shearing2 his sheep in Carmel.
Now the name of the man [was] Nabal; and the name of his wife Abigail: and [she was] a woman of good understanding, and of a beautiful countenance: but the man [was] churlish and evil in his doings; and he [was] of the house of Caleb.
And David heard4 in the wilderness that Nabal did shear6 his sheep.
And David sent4 out ten young men, and David said4 unto the young men, Get you up3 to Carmel, and go1 to Nabal, and greet1 7965 him in my name:
And thus shall ye say1 to him that liveth [in prosperity], Peace [be] both to thee, and peace [be] to thine house, and peace [be] unto all that thou hast.
And now I have heard1 that thou hast shearers:6 now thy shepherds6 which were with us, we hurt52 them not, neither was there ought missing8 unto them, all the while they were in Carmel.
Ask3 thy young men, and they will shew55 thee. Wherefore let the young men find4 favour in thine eyes: for we come1 in a good day: give,3 I pray thee, whatsoever cometh4 to thine hand unto thy servants, and to thy son David.
And when David's young men came,4 they spake17 to Nabal according to all those words in the name of David, and ceased.4
And Nabal answered4 David's servants, and said,4 Who [is] David? and who [is] the son of Jesse? there be many1 servants now a days that break away102 every man from his master.
Shall I then take1 my bread, and my water, and my flesh that I have killed1 for my shearers,6 and give1 [it] unto men, whom I know1 not whence they [be]?
So David's young men turned4 their way, and went again,4 and came4 and told55 him all those sayings.
And David said4 unto his men, Gird ye on3 every man his sword. And they girded on4 every man his sword; and David also girded on4 his sword: and there went up4 after David about four hundred men; and two hundred abode1 by the stuff.
But one 5288 of the young men told52 Abigail, Nabal's wife, saying,2 Behold, David sent1 messengers out of the wilderness to salute15 our master; and he railed4 on them.
But the men [were] very good unto us, and we were not hurt,90 neither missed1 we any thing, as long as we were conversant98 with them, when we were in the fields:
They were a wall unto us both by night and day, all the while we were with them keeping6 the sheep.
Now therefore know3 and consider3 what thou wilt do;4 for evil is determined1 against our master, and against all his household: for he [is such] a son of Belial, that [a man] cannot speak15 to him.
Then Abigail made haste,17 and took4 two hundred loaves, and two bottles of wine, and five sheep ready dressed,7 and five measures of parched [corn], and an hundred clusters of raisins, and two hundred cakes of figs, and laid4 [them] on asses.
And she said4 unto her servants, Go on3 before me; behold, I come6 after you. But she told52 not her husband Nabal.
And it was [so, as] she rode6 on the ass, that she came down6 by the covert of the hill, and, behold, David and his men came down6 against2 her; and she met4 them.
Now David had said,1 Surely in vain have I kept1 all that this [fellow] hath in the wilderness, so that nothing 3972 was missed8 of all that [pertained] unto him: and he hath requited55 me evil for good.
So and more55 also do4 God unto the enemies6 of David, if I leave55 of all that [pertain] to him by the morning light any that pisseth56 against the wall.
And when Abigail saw4 David, she hasted,17 and lighted4 off the ass, and fell4 before David on her face, and bowed101 herself to the ground,
And fell4 at his feet, and said,4 Upon me, my lord, [upon] me [let this] iniquity [be]: and let thine handmaid, I pray thee, speak17 in thine audience, and hear3 the words of thine handmaid.
Let4 not my lord, I pray thee, regard this man of Belial, [even] Nabal: for as his name [is], so [is] he; Nabal [is] his name, and folly [is] with him: but I thine handmaid saw1 not the young men of my lord, whom thou didst send.1
Now therefore, my lord, [as] the LORD liveth, and [as] thy soul liveth, seeing the LORD hath withholden1 thee from coming2 to [shed] blood, and from avenging53 thyself with thine own hand, now let thine enemies,6 and they that seek18 evil to my lord, be as Nabal.
And now this blessing which thine handmaid hath brought52 unto my lord, let it even be given8 unto the young men that follow102 7272 my lord.
I pray thee, forgive3 the trespass of thine handmaid: for the LORD will certainly2 make4 my lord a sure12 house; because my lord fighteth12 the battles of the LORD, and evil hath not been found11 in thee [all] thy days.
Yet a man is risen4 to pursue2 thee, and to seek15 thy soul: but the soul of my lord shall be bound7 in the bundle of life with the LORD thy God; and the souls of thine enemies,6 them shall he sling out,17 [as out] of the middle 3709 of a sling.
And it shall come to pass, when the LORD shall have done4 to my lord according to all the good that he hath spoken14 concerning thee, and shall have appointed14 thee ruler over Israel;
That this shall be no grief unto thee, nor offence of heart unto my lord, either that thou hast shed2 blood causeless, or that my lord hath avenged53 himself: but when the LORD shall have dealt well52 with my lord, then remember1 thine handmaid.
And David said4 to Abigail, Blessed7 [be] the LORD God of Israel, which sent1 thee this day to meet2 me:
And blessed7 [be] thy advice, and blessed7 [be] thou, which hast kept1 me this day from coming2 to [shed] blood, and from avenging53 myself with mine own hand.
For in very deed, [as] the LORD God of Israel liveth, which hath kept me back1 from hurting53 thee, except thou hadst hasted14 and come4 to meet2 me, surely there had not been left8 unto Nabal by the morning light any that pisseth56 against the wall.
So David received4 of her hand [that] which she had brought52 him, and said1 unto her, Go up3 in peace to thine house; see,3 I have hearkened1 to thy voice, and have accepted4 thy person.
And Abigail came4 to Nabal; and, behold, he held a feast in his house, like the feast of a king; and Nabal's heart [was] merry within him, for he [was] very drunken: wherefore she told52 him nothing, less or more, until the morning light.
But it came to pass in the morning, when the wine was gone out2 of Nabal, and his wife had told55 him these things, that his heart died4 within him, and he became [as] a stone.
And it came to pass about ten days [after], that the LORD smote4 Nabal, that he died.4
And when David heard4 that Nabal was dead,1 he said,4 Blessed7 [be] the LORD, that hath pleaded1 the cause of my reproach from the hand of Nabal, and hath kept1 his servant from evil: for the LORD hath returned52 the wickedness of Nabal upon his own head. And David sent4 and communed17 with Abigail, to take2 her to him to wife.
And when the servants of David were come4 to Abigail to Carmel, they spake17 unto her, saying,2 David sent1 us unto thee, to take2 thee to him to wife.
And she arose,4 and bowed101 herself on [her] face to the earth, and said,4 Behold, [let] thine handmaid [be] a servant to wash2 the feet of the servants of my lord.
And Abigail hasted,17 and arose,4 and rode4 upon an ass, with five damsels of hers that went6 after her; and she went4 after the messengers of David, and became his wife.
David also took1 Ahinoam of Jezreel; and they were also both of them his wives.
But Saul had given1 Michal his daughter, David's wife, to Phalti the son of Laish, which [was] of Gallim.
SpeedBible Software © 2001-2002 by johnhurt.com


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For All Points-Of-The-View.
A Last Love Letter To My Dad, Roosevelt, (Rosie), Holman. From Sandy Lynne Holman, who you lovingly called "Knot Head" See Video Tribute on you tube link. Fly with the Angels Dad. I love you endlessly. (https://youtu.be/vo0ftmqdD7s)
When I felt that divine voice within me saying to take the whole week off, which started with Fathers Day in June 2017, and spend evenmore time with you I did not question it. I have learned to listen to Gods' promptings in life. Fathers Day was beautiful but I sensed it would be my last one with you. When I had that dream from God a few days later where Mom was waiting for you in white smiling the biggest smile I had ever seen, and you were in white with a new hair do, smiling back at her and asking if she approved, I knew it was from God. Mom, without moving her lips, let me know you would be coming home soon. I told your Son In Law, who you affectionately called the "Captain and Toni, one of your other daughters about the dream. I had hoped we might make it to your birthday, which was a couple months, away, but you transitioned a few days later. I am thankful to God for that prophetic dream because it helped me when the angels came to take you home. It helped me not to be absolutely crushed since our love was an extraordinary, abundant agape love for each other and existed in rare air. You gave all your family and loved ones this kind of transcendent love and it was felt in every part of our bodies,minds, and souls. I felt it in the depths of my spirit and it carried me through many tough times and gave me "other worldly" joy.
Dad, I will miss you something terrible yet I have no regrets. We loved on each other tremendously, spent priceless times together and shared countless belly laughs in each other's company. The kind that make you bend over. We had intimate conversations about almost everything and nothing was off limits. It helped me truly get to know you and your incredible massive loving heart. When Grandpa Rufus, your father, died our relationship even went deeper, if that were even possible, and I felt my soul and spirit merge with you in divine and profound ways. I learned that I was truly your daughter at the deepest levels and that your imprint was all over me. I loved on peoplelike you, gave suffocating bear hugs, celebrated life at the highest levels, laughed so hard that you could not breathe and even walked like you. I had your powerful gift of discernment with people and your phenomenal analytical mind. You taught me to see through people and to get a sense of whom they really are and this served me very well in life. Any tough bone I have within me is truly from you and the wisdom you gained growing up on the tough streets ofDetroit. This kept me from being a "door mat" since I had Moms "love everyone inclinations." I call everyone sweetheart just like you and still do. You also called people, doll, doll face, monkey and of course I was your precious "knot head" and I loved hearing you call me that name. I still hear you saying it in my soul when I think about you. I was the luckiest person in the world to have come from Grandpa, You and Mom and the three of you impacted me more than anyone outside of God. It is so clear that the essence of all of you is seared in my soul and every part of my being. I am privileged to have come to this world through you and will honor your memories forever by living the best life I can and using my gifts to make a difference. Thank you Dad for modeling true love, brilliance, adventure, a life of no regrets, charity, perseverance, toughness, being you, creativity, and the power of a big bear hug. Thanks for being the biggest hero, (including Mom and Grandpa), in my life outside of God. I will forever carry you all, as my family ancestors, in every fabric of my being. Rest in peace Royal Court and give my sister Cherie a big hug who loved on me deeply too. You will be missed but we will see you again. To God is the Glory!
A poem from your daughter Sandy Holman, Knot head
Thanks to my brothers and Sisters for Taking Care of my Father at different times in his living. I love you all and Dad did too/ Sandy Lynne Holman, July 6th, 2017: Rosie Holman's' Home Going Services
Sweet Heart – Baby Doll- knot head, a Juicy Kiss
These are just a few things that his loved ones will miss
Dad was genuine, discerning, and full of Motown Style
He made all of us feel like we were his favorite Child
He lived a full life and it was as good as it gets
How many people die saying that they have no regrets
He had God, love, a Family and Friends
These are the things that matter most in this life we are in
He taught all of his family, key lessons for life
And would say something like "Baby you have to learn to throw out all of that strife
He was determined, positive, a reflection of his Mom
That is why when others lost it, our Dad could stay calm
Don't get me wrong though you were in trouble, if you saw him biting his lip
It meant you messed up and would get his corrective wit
But you always knew he loved you, he always gave you that smile
And you knew every thing would be ok after just a little while
We loved that he modeled living his dream
Be it work, bowling, or playing with a baseball team
He loved boxing, fishing, and golfing too
And He made you feel special when he hung out with you
He adored the temptations and all the soulful sounds
He could sing like a professional, Dad could really get down
He loved Lake Tahoe, playing cards and traveling too
Dad believed you should do all the things that you wanted to do
How do you measure the true worth of amazing man?
You look at his heart and the love he had within
A person may not be perfect, but for all of us this is true
But it took just one big bear hug to feel the love Dad had for you
He was brilliant, a jesser, looking cool and awesome to meet
He didn't care if you were rich or a person who lived on the street
He said his parents were the reason that he lived a long time
They taught him about respect and told him "he was just fine!"
He was told to treat others with charity and concern
And that everyone always had something else they could learn
If our father where here and watching all of you.
He would be so touched and the tears would start coming through
He would take a deep pause and after a little time
He would say, "Don't worry about me," I am doing just fine
No need to be sad or have crocodile tears for me
You Dad loves God and, my spirit is free
Dad loved all of his Family with every bit of his heart
God will be with you forever, just go do your part
Be who you are and go live out your hearts desire
Forgive and love others and always try to inspire
Don't let anyone label you are keep upset and down
And know without doubt you have a warrior angel now
Love you Baby doll sweetheart, monkey and knot head.
Dad is spirit now, only the body is dead
Love you Rodney, Greg, Toni, Michelle and Ricci
Cherie knows I love her and she is up here with me
Love you grand kids and my great grandchildren too
Always know that Pa Pa will be up here watching over you…
To my wife, adopted children, friends and extended kin
You can't imagine the space that I have just entered in………
The angels came softly to transport me home,
God made sure your Daddy would not be alone
I lived life fully and in my own special way
Gods' love wrapped around me on that divine Sunday
Sweet precious Abba, please carry my family through
And Have your holy spirit guide them in all that they do
Embraced by your presence and grace, when I took flight that night
I now bask in your glory and honor your light………
Our father, who art in heaven................................... I love you too Knot head J
We all love you Dad with all our hearts. Thanks for loving us all and modeling how to live a full life. Be at peace with God…
Sandy Holman, July 4th, 2017. Dad died June 25th and 9; 45 pm a week after fathers day on Sunday……….
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This week, President Barack Obama issued a plan to green the federal government's fleet of vehicles—specifying that by 2015, 100 percent of its vehicles purchased would be clean-energy, fuel-efficient models. Today officials confirmed that's not quite true; it applies to all but the...Read More»
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Northern Colorado car owners and legislators have been grumbling over the impact of the requirement to have vehicles tested for emissions. This is new to the vehicle screening regime in Larimer and Weld counties, and would require vehicles to go through testing similar to what is done in the Denver...Read More»
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Looking ahead to spring and summer – maybe even fall – for some of the new cars debuting at this year's winter auto shows? Continuing our two-part story on hotly-anticipated new cars, FamilyCarGuide takes a look at five more products from automakers that we can't wait to sit...Read More»
Transmissions are anything but boring these days. Witness the furious development fever going on to maximize power while at the same time increase fuel efficiency in today's cars, trucks, SUVs, CUVs, and minivans. Years ago it was four-speed or nothing. Then six-speed trannies became vogue...Read More»
Companies are always trying to spiff up their images. Whether they sell cars, clothing, or fast food, businesses work hard to make consumers believe they stand for something bigger than merely "selling things". Chevrolet's newest online initiative aims to do that by aligning the brand with Dr...Read More»
The refreshed 2011 Chrysler 300 sedans might not look that much different from their predecessors on the outside, but inside you'll see a difference. In addition to a new base engine and extensively upgraded appointments for what's essentially an all-new cabin, the Chrysler 300 is set to wow with...Read More»
Shopping for a new car? The old, reliable laws of supply and demand could do you a big favor, even if you're looking for a high-performance car--if you plan right. It's good to be the only one who wants something. And in the current economy, few people seem to want high-performance cars. Yes, we've...Read More»
We've seen the 2011 Chrysler 300 in the company's Chapter 11 filing, but snippets are landing on the Interwebs like the first flakes of winter. Is it time already? [Jalopnik] While Chrysler divorces the Ram brand from Dodge, it's giving the fledgling nameplate its own iPhone app. Perfect for the...Read More»
Toyota recalls, the electric-car race, and the 2012 Chrysler 300 -- it's all here today at High Gear Media: The 2011 Ford F-150 gains four new engine options, everything from a base V-6 to a turbo EcoBoost six and a pair of V-8s. Pickups are back...and better than ever. [TheCarConnection] Can one...Read More»
Chrysler today announced a more aggressive incentives program for May, including the potential for no-interest financing on much of its product line or cash-back offers of up to $4,000. For up to 36 or 60 months, depending on the vehicle, Chrysler is offering zero-percent financing, arranged...Read More»
The results of Chrysler's first arbitration hearing are in, and the news for dealers isn't good. The arbitrator has determined that Joe Kidd Dodge of Cincinnati, Ohio met Chrysler's criteria for elimination and that Chrysler broke its contract with the dealer in an impartial manner. As Chrysler...Read More»
The past couple of years haven't been kind to automakers. Like ballast on a sinking ship, many brands were thrown overboard as car companies scrambled to stay afloat. One major automaker managed to dodge that kind of unpleasantness, but now a major auto analyst thinks it's high time for a purge...Read More»
Article by Bruce Richter; photographs by Robert Didal. The 2010 New York Auto Show had several themes this year. One was the prevalance and prominence of the greenest solutions, with literally dozens of hybrid and electric vehicles, which I covered on AllCarsElectric.com. The show's second...Read More»
After a very long dry spell, the good news for Chrysler fans is finally flowing again. First we heard that the automaker was ratcheting up its production timeline on the eagerly anticipated 2011 Chrysler 300. Then, Fiat mentioned that it's on-track to assume a 35% stake in Chrysler within the next...Read More»
In the past, consumers only really had two drivetrain choices, RWD (rear wheel drive) and 4WD (4-wheel drive). Passenger cars and trucks were driven by the rear wheels, and a few specialty trucks got the 4-wheel drive option. Now it seems hard to find a RWD sedan, and the lines between 4WD and...Read More»
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Over the past few months, we've seen some curious ads for Chrysler. There was the "My Name Is Ram" clip, which was endlessly parodied on YouTube. Then there was that spot for the Chrysler 300, starring Mikhail Gorbachev, Nelson Mandela, and a plea for Aung San Suu Kyi's freedom. And this Monday, we...Read More»


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We Need Compromise Not Knife Fights
11 months ago Citizen#7 0
Democrats and Republicans are at each others throats daily with their own set of facts and position they protect at all costs. The losers in these daily battles are average citizens be they Democrat or Republican.
Right now the Democratic Party is the underdog, eight years ago it was the Republican party who was the underdog. Back then Democrats rolled back what the Republicans did. Today Republicans are busy planning to undo what the Democrats did. Where will we end up, probably about where we started, with little change.
Both parties don't seem to be all that different. They promise lots, deliver little, and blame the other side for the problems.
We've become so polarized by "Alternate Facts", "Fake News", and a 24/7 news media dramatizing the differences rather than similarities between us to gain something called "market share" which translates to money for the network. I for one am tired of "Breaking News" that often is far from important.
The elected elites in both parties have almost to a person taken money from lobbyists and PACS. Much of what they "know" about issues comes from lobbyists representing one side or the other of an issue. All but a few politicians play fast and loose with the truth. We citizens have allowed them to get away with lying, stealing, and ignoring average citizens. We've accepted this shabby treatment as "that's the way it is".
What we need is a political system that puts the welfare of average citizens above party, above PAC's and lobbyists, and above the one percent. We need our elected officials to focus on whats best for the country not for any one politician or political party. The division that exists today in politics serves the rich and powerful. The more fighting and polarization that occurs the more the rich and powerful win and we lose. They win access to maintain the status quo or get the changes in the most powerful country in the world that benefits them.
We've lost an appreciation of compromise as a strength not a weakness. We have real issues like stagnant wages, job loss, enemies that wish us harm, and much more. What we don't need are simple slogans and speeches that defy common sense. We don't need to be placated with half truths and lies, we need our leaders to roll up their sleeves and honestly tackle the work that needs to be done.
To many times important work is tied with expensive strings that multiply costs many times and yield questionable benefits. Often the "important work / legislation" costs multiple times more than it should. We working class citizens are left to pay the bill while the elected elite pat themselves on the back for a job "well done". They are rewarded with increasing amounts of money to fill campaign coffers, and support lavish lifestyles.
Saul Alinsky in 1971 published a book called, "Rules for Radicals A Pragmatic Primer for Realistic Radicals".
In his book Alinsky wrote,
It is a grave situation when a people resign their citizenship or when a resident of a great city, though he may desire to take a hand, lacks the means to participate. That citizen sinks further into apathy, anonymity, and depersonalization. The result is that he comes to depend on public authority and a state of civic-sclerosis sets in.
We as a country have been infected with the civic-sclerosis Alinsky spoke of. George Orwell wrote in Animal Farm "All animals are equal, but some animals are more equal than others." Our political elites in both parties as well as their big money masters have adopted Orwell's Animal Farm philosophy at our expense.
Let's take a breath and challenge all of our legislators on both sides to work together and do their job, after all they work for us, not the other way around.


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Date(s) - 30/08/2013 - 02/09/2013
All Day
Washington State Convention Center
In 2004, the folks at Penny Arcade decided they wanted a show exclusively for gaming. Sure, comics, anime, and other nerd hobbies were cool, and those activities all had their own shows… so what about games? From that idea spawned a small 4,500 person event in Washington, focused on the culture and community that is gaming.
PAX Prime 2013 will be the first four-day PAX and is scheduled to occur from August 30 to September 2, 2013.
For more information, please visit: http://prime.paxsite.com


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Set your summer intention: NOW!
Posted On June 21, 2013 By Karen Fabian And has No Comment
Summer is my favorite season. Take a moment today and set some intentions for your summer. It's one of those seasons that seems so short.. we wait through the long winter and then it seems to fly by. Take a moment today and make a list of the things you'd like to do. Then, save your pennies, plan your schedule and make this the best summer ever!


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It's been six years since my beautiful Cherry kitchen cabinets were installed. Since then, the bottoms have fallen out of the 36″ wide drawers. Twice. The first time the company came out to inspect and sent replacement boxes. The second time, Paul rebuilt the boxes for me to make sure they would last.
He delivered the silverware drawer to me on Friday. As I started to put all of the silverware back in the drawer, I told him that I needed dividers. And in a perfect world, I'd have nice, neat, built-in dividers.
Paul said if he just had the right kind of wood, he could "fix me right up."? I took that opening and disappeared into the garage. After bit of digging around behind boxes in a forgotten corner, I found a piece of thin plywood left over from some other project and delivered that to him with a pencil sketch of what I wanted.
Now that he had to put up he said, "Sweetie, I'm going to rock your world."
And he did. Built in custom drawer dividers. I'm in heaven. Am I easy or what?


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Anti-cancer drugs that disrupt mitosis inhibit cell proliferation and induce apoptosis, although the mechanisms of these responses are poorly understood. Here, we characterize a mitotic stress response that determines cell fate in response to microtubule poisons. We show that mitotic arrest induced by these drugs produces a temporally controlled DNA damage response (DDR) characterized by the caspase-dependent formation of γH2AX foci in non-apoptotic cells. Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression. We show that this response is controlled by Mcl-1, a regulator of caspase activation that becomes degraded during mitotic arrest. Chemical inhibition of Mcl-1 and the related proteins Bcl-2 and Bcl-xL by a BH3 mimetic enhances the mitotic DDR, promotes p53 activation and inhibits subsequent cell cycle progression. We also show that inhibitors of DDR protein kinases as well as BH3 mimetics promote apoptosis synergistically with taxol (paclitaxel) in a variety of cancer cell lines. Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies.
2. Introduction
Microtubule poisons such as taxol (paclitaxel) disrupt mitotic spindle assembly and delay or arrest proliferating cells in a prometaphase-like state through the prolonged activity of the spindle assembly checkpoint (SAC), which restrains anaphase by inhibiting the widespread destruction of mitotic regulators such as cyclin B1 and securin [1]. Cells held in mitosis by microtubule poisons can undergo caspase-dependent apoptosis [2], which is influenced by the duration of the mitotic arrest [3,4], or they can slip out of mitosis after a prolonged period [5,6]. Premature exit from mitosis usually decreases sensitivity to microtubule poisons [4], consistent with the generation of apoptosis-promoting activity during mitotic arrest. Apoptosis can still occur, however, after exit from mitosis [3,6]. The balance between these cell fates is likely to determine the effectiveness of microtubule poisons and other anti-mitotic drugs in cancer chemotherapy [3,4,6–9].
Control of apoptosis during mitosis is determined by protein phosphorylation and the timed, ubiquitin-mediated destruction of key regulators of the intrinsic apoptotic pathway by the proteasome [10]. If metaphase is not successfully resolved, then apoptosis can be initiated after the anti-apoptotic protein Mcl-1 is degraded, which occurs subsequent to its phosphorylation by CDK1–cyclin B1 [11,12]. In addition, the related anti-apoptotic proteins Bcl-2 and Bcl-xL are phosphorylated and might be inhibited during mitotic arrest [13]. The slow degradation of cyclin B1 even though the SAC is active can lead eventually to release from mitosis [5]. Whether or not apoptosis is initiated during the arrest or after a cell enters interphase is thought to depend upon the relative timing of changes in the activities of CDK1–cyclin B1 and regulators of apoptosis during mitotic arrest [2,6,10].
A prolonged delay in mitosis can also determine subsequent cell proliferation [14]. However, the nature of the putative mitotic stress signal and the mechanism by which delayed mitosis can influence subsequent cell cycle progression have been unclear. Recent research has shown that there is an accumulation of DNA damage in cells arrested in mitosis, as evidenced by an increase in phosphorylated histone H2AX (γH2AX) [15,16], and the transcription factor p53 is induced after release into interphase [14,15,17]. Evidence has also been provided that mitotic arrest can cause DNA damage owing to partial apoptosis [17]. These findings suggest that stress responses initiated by mitotic disruption can determine subsequent cell cycle progression and that this process might be related to the mechanism of apoptosis.
Here, we show that a prolonged delay in mitosis initiates a time-dependent DDR that controls subsequent cell fate through the activation of protein kinases and p53. This pathway is dependent on subapoptotic caspase activity and is controlled by Bcl-2 family proteins. Thus, we identify a potential novel mechanism for the oncogenic activity of Bcl-2 family proteins through indirect control of the cell cycle and apoptosis. We also demonstrate that pharmacological manipulation of Bcl-2 family proteins or DDR kinases enhances the cell-killing effects of anti-mitotic drugs on proliferating cancer cells by enhancing the response to mitotic stress, providing a rationale for combination chemotherapy.
3. Material and methods
3.1. Reagents and antibodies
Reagents were purchased from Sigma-Aldrich unless otherwise specified. The origins of primary antibodies and the dilutions used in this study are described in the electronic supplementary material, table S1. Secondary antibodies used for Western blot and immunofluorescence analyses were from Biorad and Molecular Probes, respectively.
3.2. Cells
U2OS (HTB96) cells were obtained from Cell Services, Cancer Research UK London Research Institute. U2OS cells in which p53 was stably depleted by shRNA (pRS-p53) and control cells transfected with a scrambled shRNA (pRS-sc) [18] were a gift from K. Ryan (Beatson Institute for Cancer Research, Glasgow, UK). All the other cell lines were purchased from ATCC (LGC Standards, Teddington, UK).
3.3. Cell synchronizations and treatments
U2OS cells were synchronized in mitosis by collection of rounded-up cells after a 2 h treatment with a microtubule poison and then either replated with the drug to maintain mitotic arrest or released in fresh media. Untreated mitotic cells were obtained by washing-off asynchronous cultures. Synchronization at the G1/S boundary by double thymidine block was performed as described [11]. G1 phase cells were obtained by centrifugal elutriation [19]. U2OS cells were transfected with Mcl-1 subcloned into pCMV-Flag (MRC Protein Phosphorylation Unit, Dundee) using Superfect transfection reagent (Qiagen). Mcl-1 was depleted in U2OS cells by the siRNA GGACUUUUAGAUUUAGUGA (MWG) using RNAiMax (Invitrogen) as transfection reagent. Cells were treated as indicated with z-VAD-fmk (Enzo Life Sciences), Obatoclax/GX15–070, Navitoclax/ABT-263, NVP-BEZ235 (Selleck Chemicals) or KU55933, NU7441, SB218078 (Tocris).
3.4. Flow cytometry
One- and two-dimensional cell cycle analyses, apoptosis determined by fluorochrome-labelled inhibitor of caspases (FLICA, FAM-DEVD-fmk, Immunochemistry Technologies) and immunofluorescence analyses by flow cytometry were performed according to manufacturers instructions or as described previously [11] and detailed in the electronic supplementary material.
3.5. Western blot analyses
Protein extracts were prepared using a standard RIPA protocol and analysed as described in the electronic supplementary material.
3.6. Proliferation assays
Cell viability was monitored in an IncuCyte ZOOM (Essen) according to the manufacturer's instructions or by a standard crystal violet assay (see the electronic supplementary material for details). Synergies between drugs were determined according to the Chou–Talalay method [20] by using a crystal violet-based assay detailed in the electronic supplementary material.
3.7. Immunofluorescence assays and microscopy
γH2AX immuno-detection was performed according to a standard protocol detailed in the electronic supplementary material.
3.8. Figures and statistics
Statistical analyses and regressions were conducted using Graphpad Prism. Differences between samples were assessed using non-parametric Mann–Whitney or one-way ANOVA statistical tests as required. Figures were generated using Microsoft Excel, Graphpad Prism, Adobe Photoshop and Adobe Illustrator.
4. Results
4.1. Mitotic delay induced by microtubule poisons activates DNA damage signalling and affects subsequent cell cycle progression and apoptosis
To study cellular responses to mitotic arrest, we used a cell culture model in which rounded-up mitotic human osteosarcoma cells (U2OS) were collected after treatment of an asynchronous culture with nocodazole for 2 h (denoted N2M; figure 1a; electronic supplementary material, figure S1A). Mitotic cells were then replated in nocodazole for a further period and analysed by Western blotting (figure 1b). This procedure allows analysis of biochemical changes during the period of mitotic arrest and after subsequent entry into interphase without interference from cellular stresses generated by other methods of pre-synchronization. We found that a prolonged mitotic delay of up to 10 h followed by slippage into interphase (confirmed by flow cytometry; electronic supplementary material, figure S1B) activated components of the DNA damage response (DDR) [21] in a time-dependent manner (figure 1b). Ataxia–telangiectasia-mutated (ATM), a large phosphatidylinositol-3-kinase-related protein kinase (PIKK), became phosphorylated at Ser1981, a site associated with its activation and stabilization at sites of DNA damage [22]. The phosphorylation of ATM increased and persisted as cells slipped out of mitosis after 10 h (indicated by the loss of cyclin B1 and histone H3 Ser10 dephosphorylation), and correlated with activating phosphorylation of its targets, the protein kinase Chk2 and p53; there was also a transient activating phosphorylation of Chk1 at Ser345, a site targeted by ATR (ATM- and Rad3-related).
We confirmed that histone H2AX phosphorylated on Ser139 (γH2AX), a site targeted by ATM and the related kinases ATR- and DNA-dependent protein kinase (DNA-PK), was increased during mitotic arrest and elevated further after slippage into interphase. We also observed a strong increase upon exit from mitosis of the protein levels of both p53 and its transcriptional target, the p21waf1 cyclin-dependent kinase inhibitor (figure 1b). Together, these results confirm that a prolonged delay in mitosis initiates a primary DDR that is fulfilled in the subsequent interphase after cells have slipped out of mitosis.
We also analysed cells released from a transient mitotic arrest by washing out nocodazole after 2 h of treatment (figure 1c). Released cells (denoted N2M + 4hR) exhibited activation of the DDR in a similar fashion to cells that had slipped out of mitosis despite the continued presence of the drug (N10S). Flow cytometry confirmed that released cells underwent cytokinesis to produce diploid G1 cells with a 2N DNA content, whereas slipped cells gave rise to a tetraploid (4N) G1 population owing to failure to undergo cytokinesis (figure 1d). Thus, the secondary DDR induced after a prolonged delay in mitosis is not dependent on whether or not cells exit mitosis aberrantly and become tetraploid. In the corresponding population of floating cells that had not slipped out of mitosis (N10M; figure 1c), there was a strong increase in γH2AX that is likely to be due to widespread DNA fragmentation in apoptotic cells that accumulated during mitotic arrest (figure 1e). The caspase inhibitor zVAD-fmk blocked the generation of such apoptotic cells as expected (figure 1f). This is consistent with the ability of some mitotically arrested cells to proceed into apoptosis without first entering interphase [3,6].
In contrast to G1 cells previously arrested in mitosis, elutriated G1 cells did not exhibit any induction of the DDR (electronic supplementary material, figure S1E). Furthermore, we did not detect any effect of nocodazole or taxol on the phosphorylation or protein levels of ATM, Chk2, γH2AX, p53 or p21waf1 in cells treated only during interphase (electronic supplementary material, figure S2A,C). Irrespective of the effects of the microtubule poisons, we observed a transient increase in the activating phosphorylation of Chk1 in interphase cells progressing through G1 into S-phase, consistent with a role for this kinase in the normal control of DNA replication (electronic supplementary material, figure S2B,D). We also found that interphase cells synchronized by a double thymidine block exhibited a significant induction of γH2AX (electronic supplementary material, figure S2C; 0 h samples), which is attributable to DNA replication stress. Because some of this damage might persist into mitosis, we avoided the further use of this protocol to study mitotic responses.
4.2. The period of mitotic arrest controls DNA damage signalling, cell cycle progression and apoptosis
To test whether the period of mitotic arrest affects subsequent responses, we maintained cells for either 2 or 6 h in nocodazole-induced mitotic arrest, then released the cells by washing out the drug and analysed the DDR. Cells that had been arrested in mitosis for a total of 6 h exhibited stronger activating phosphorylation of ATM, Chk2 and p53 after release from mitosis and they were more likely to be delayed in G1 phase (BrdU negative adherent cells with 2N DNA content) than those that had been arrested in mitosis for only 2 h (figure 2a,b). The activating phosphorylation of Chk1 was also prolonged (figure 2a), consistent with a delay in S phase initiation (figure 2b). Similar results were obtained when cells were treated with taxol rather than treated with nocodazole (electronic supplementary material, figure S3A,B). Thus, the period of mitotic arrest determines the intensity of the DNA damage signalling and this correlates with restraint of subsequent cell cycle progression. Moreover, we found that U2OS cells arrested for longer in mitosis died more readily by apoptosis after release into interphase (figure 2c and electronic supplementary material, figure S3C). Together, these effects on cell cycle progression and apoptosis resulted in the strong inhibition of cell proliferation (figure 2d; electronic supplementary material, figure S3D).
4.3. The DNA damage response in mitotic cells is caspase-dependent and is regulated by Bcl-2 family proteins
In agreement with previous descriptions [15,16], we found that most cells arrested in mitosis for 2 or more hours exhibited localized γH2AX foci (figure 3). Counting the number of γH2AX foci, we found that untreated mitotic cells exhibited only occasional foci (figure 3a) with a mean of 2.2 foci per cell (figure 3b). By contrast, cells arrested in mitosis had a distribution of up to 20 foci per cell (electronic supplementary material, figure S4), with a mean of 5.4 in cells arrested for 2 h. The mean number of foci per cell increased when the period of the arrest was prolonged, showing that it is time-dependent (figure 3b; electronic supplementary material, figure S4). Importantly, we found that the formation of these foci was inhibited by the caspase inhibitor zVAD-fmk (figure 3). By contrast, γH2AX foci induced in mitotic cells by the topoisomerase II poison etoposide, which causes widespread DNA double-strand breaks, were not dependent on caspase activity (electronic supplementary material, figure S5).
Mcl-1 is gradually degraded during a prolonged mitotic arrest [11,12], but ectopic expression of Mcl-1 inhibited the formation of γH2AX foci in cells arrested in mitosis. Conversely, the Mcl-1/Bcl-2/Bcl-xL inhibitor Obatoclax (GX15-070) [23] (figure 3b; electronic supplementary material, S4B) or siRNA-mediated ablation of Mcl-1 expression (figure 3c) increased the number of γH2AX foci per cell. These results demonstrate that many of the γH2AX foci formed during mitotic arrest require caspase activity and are under the control of Mcl-1, and possibly other related regulators of caspase activation. The distribution of the number of foci per cell (electronic supplementary material, figure S4B) suggests a stochastic element in the mechanism initiating foci damage.
4.4. Mcl-1 controls the DNA damage response and p53 activation following a delayed mitosis
Analysis of the DDR in interphase cells following release from mitotic arrest (figure 4a) showed that the activating phosphorylation of ATM, Chk1, Chk2 and p53, as well as the induction of p21, were strongly inhibited by addition of zVAD-fmk during the arrest (figure 4b, left panel), and were therefore dependent on upstream caspase activity. This is consistent with a mechanism in which the response to caspase-dependent DNA damage foci generated during a delayed mitosis is enhanced following release into interphase. By contrast, the increase in total p53 protein following mitotic delay was not affected by zVAD-fmk, indicative of a caspase-independent mechanism for p53 induction. Caspase-dependent cleavage of poly(ADP ribose) polymerase (PARP) was not observed until 10 h of incubation (N10M), consistent with flow cytometry data (figure 1e) showing that, until this time, few cells were undergoing apoptosis. Remarkably, however, treatment with zVAD-fmk reduced the delay in G1 of non-apoptotic cells released from 2 h mitotic arrest and promoted their entry into S phase (figure 4c, left panel).
The amount of Mcl-1 was diminished more markedly by 6 h mitotic arrest than by 2 h mitotic arrest, and this loss was not affected by zVAD-fmk (figure 4b, left panel), in agreement with the caspase-independent degradation of this protein in an APC/C-dependent manner during mitotic arrest [11]. Ectopic expression of Mcl-1 strongly inhibited the DDR (figure 4b, middle panel) and also promoted subsequent entry into S phase of cells that had been delayed in mitosis (figure 4c, middle panel). Conversely, inhibition of Mcl-1 and the related anti-apoptotic proteins Bcl-2 and Bcl-xL by Obatoclax enhanced the DDR (figure 4b, right panel) and delayed cells in G1 (figure 4c, right panel), whereas no significant apoptotic cleavage of PARP was detected (figure 4b, right panel). Neither ectopic Mcl-1 expression nor Obatoclax treatment affected the induction of p53 protein following mitotic arrest, but they did strongly inhibit or enhance, respectively, the phosphorylation of p53 at the activating site serine 15, and there was a corresponding effect on the induction of p21. Thus, activation of the p53 pathway by DNA damage signalling after mitotic delay, with the subsequent regulation of cell cycle progression, requires caspase activity and is under the control of Mcl-1, and possibly other Bcl-2 family proteins.
4.5. The role of p53 in the fate of cells following mitotic delay
Because mitotic delay resulted in the activation of p53 downstream of DNA damage signalling, we tested the role of p53 in the cellular response to a synchronized mitotic delay (figure 5). We examined the effects of mitotic arrest on a cell line derived from U2OS cells [18] in which p53 was stably depleted by expression of an shRNA (pRS-p53); cells expressing a scrambled shRNA (pRS-sc) were used for comparison. Depletion of p53 inhibited the induction of p21 following arrest in mitosis by nocodazole for 2 h (figure 5a) and prevented the accumulation of cells in the subsequent G1 phase (figure 5b). By contrast, loss of p53 did not affect the normal cell cycle progression of mitotic cells collected without nocodazole treatment. Interestingly, the rate of progression into S-phase (2N-4N BrdU positive cells) was apparently enhanced in p53-depleted cells by prior mitotic delay as compared with those cells that had progressed from a normal mitosis (figure 5b). These results demonstrate that p53 plays a key role in restraining cell cycle progression following mitotic stress.
Depletion of p53 also caused a significant increase in apoptotic caspase-3 activity measured 48 h after release from mitotic arrest (figure 5c). p53 therefore has an anti-apoptotic, pro-survival function following mitotic stress. Nevertheless, depletion of p53 had no overall effect on the number of cells that accumulated after release from mitosis, even though the effect of period of mitotic arrest was apparent (figure 5d). This suggests that, in this cell type, the lack of restraint on cell cycle progression in the absence of p53 is counteracted by an increase in apoptosis, so that overall cell proliferation is inhibited to the same extent by mitotic stress whether or not the cells can activate p53. Qualitatively similar results were obtained in experiments using either nocodazole (figure 5) or taxol (electronic supplementary material, figure S6) to induce mitotic arrest, although cells arrested using taxol were more prone to subsequent apoptosis or inhibition of cell proliferation than cells arrested for the same period with nocodazole (electronic supplementary material, figures S3 and S6).
4.6. Inhibition of DNA damage response kinases enhances the effects of mitotic stress
To address the significance of the DNA damage pathway activated in response to mitotic arrest, we analysed the effect of inhibiting ATM, ATR or DNA-PK after release from mitosis using the chemical inhibitors KU55933 [24], NU6027 [25] and NU7441 [26,27], respectively (for protocol, see figure 6a). Chemical inhibitors have an advantage over genetic approaches, because kinases can be targeted at specific points in the cell cycle; however, there remains the caveat that their cellular effects might be mediated by inhibition of other enzymes, especially other kinases (e.g. inhibition of CDK2 by NU6027 [25]). The relative selectivity of KU55933 and NU6027 was confirmed by their ability to block the phosphorylation of the ATM substrate Chk2 and the ATR substrate Chk1, respectively, as well as the selective inhibition of ATM autophosphorylation at S1981 by KU55933 but not NU6027 or NU7441 (figure 6b). We found that inhibition of ATM or DNA-PK in particular strongly suppressed the γH2AX signal generated immediately after release from mitosis, with a weaker effect by the ATR inhibitor. In addition, the ATM and ATR inhibitors both impaired p53 Ser15 phosphorylation and p21 induction at this time (figure 6b, left panels). We conclude that ATM, ATR and DNA-PK all contribute to DNA damage signalling after release from mitotic arrest.
Conversely, while the overall DDR had declined 24 h after release from mitotic arrest without kinase inhibitors, the γH2AX signal at this time was strongly enhanced by inhibition of ATM or ATR, and was maintained to a lesser extent by inhibition of DNA-PK. Each of the three inhibitors also induced further induction of p53 and p21 (figure 6b, right panels). This indicates that ATM, ATR and DNA-PK all play roles in recovery from the initial damage, which is prolonged when one or other is inhibited. Inhibiting each of these PIKKs reduced the ability of U2OS cells released from mitosis to enter S phase (figure 6c,d). This effect was more pronounced in cells that had been arrested in mitosis for 2 h (figure 6c) than in cells that had been synchronized in a normal mitosis (figure 6d). Furthermore, PIKK inhibitors increased the number of apoptotic cells in cultures that had been arrested in mitosis by nocodazole (figure 6e; electronic supplementary material, figure S7A,B) or taxol (electronic supplementary material, figure S7C,D) prior to release into interphase. Consistent with these effects, PIKK inhibitors restrained the proliferation of cells that had been arrested previously in mitosis (figure 6f; electronic supplementary material, figure S7B). Thus, ATM, ATR and DNA-PK each play pro-survival roles in interphase following mitotic stress, and their inhibition leads to enhanced cell cycle arrest, increased apoptosis and a reduction in overall cell proliferation.
4.7. BH3 mimetics and inhibitors of the DNA damage response act synergistically to sensitize cancer cells to microtubule poisons
We found that inhibitors of ATM, ATR and DNA-PK, including NVP-BEZ235, which also inhibits phosphatidyl-3-kinase and mTOR [28], as well as a Chk1 inhibitor (SB218078 [29]), all enhanced the induction of apoptosis in U2OS cells pre-treated for 6 h with taxol. So did Obatoclax or another BH3 mimetic Navitoclax (ABT-263) that selectively inhibits Bcl-2 and Bcl-xL [30] (figure 7a,b). Conversely, taxol strongly enhanced the induction of apoptosis by both kinase inhibitors and BH3 mimetics because these drugs caused only low levels of apoptosis in the absence of taxol. Each of the kinase inhibitors and BH3 mimetics decreased the total number of U2OS cells whether or not the cells expressed p53, although suppression of p53 reduced the inhibition of cell proliferation by taxol (figure 7c).
We analysed the proliferative response of a variety of cancer cells from the NCI-60 cell lines panel using the Chou–Talalay method [20] to assess quantitatively the synergy between drugs (see electronic supplementary material, experimental procedures). Proliferation indexes corresponding to the combination indexes (CIs) plotted in figure 7d are shown in electronic supplementary material, figure S8. We found that there was a synergistic inhibitory effect (CI < 1) of taxol in combination with either BH3 mimetics or inhibitors of DDR kinases on the proliferation of many of the cell lines (CI curves obtained in U2OS cells are given in electronic supplementary material, figure S9), although there were significant differences between them (figure 7d; electronic supplementary material, table S2). Navitoclax had a very potent inhibitory effect on the proliferation of all the cell lines, but only when combined with taxol, indicating that this may be a generally effective combination therapy. Furthermore, NVP-BEZ235 produced a very strongly synergistic inhibitory effect with taxol in all of the cell lines, including those lacking p53.
5. Discussion
In this report, we have characterized a temporally controlled mitotic stress response pathway that is regulated by the oncogenic proteins Mcl-1 and Bcl-2/Bcl-xL. We show that this pathway determines the sensitivity of cancer cells to microtubule poisons and can be manipulated by combinations of these drugs with inhibitors of Bcl-2 family proteins or DDR kinases.
Regulation of the mitotic stress response by Bcl-2 family proteins suggests a mechanism for temporal control of the initiation of the response (figure 8). Cells in mitosis are restricted in their ability to induce new transcription and changes during mitosis are mediated primarily through post-translational mechanisms, notably protein phosphorylation and protein degradation by the ubiquitin–proteasome system. Mcl-1 is destroyed after a prolonged mitotic arrest [11], and loss of Mcl-1 is likely to be a key step in the initiation of the response. Although Bcl-2 and Bcl-xL are stable during mitotic arrest, they are highly phosphorylated, and this phosphorylation has been associated with the inhibition of their function [13]. Nevertheless, cells arrested in mitosis are exquisitely sensitive to Navitoclax [31] (D.J.C., K.O.H., L.A.A. & P.R.C. 2015, unpublished data), showing that Bcl-2 and/or Bcl-xL are critical to prevent the induction of mitotic stress when Mcl-1 has been degraded. It seems, therefore, that the threshold to induce caspase activation is reduced in mitotically arrested cells through both the destruction of Mcl-1 and the partial inhibition of Bcl-2/Bcl-xL.
Subapoptotic activation of caspase-3/7 is likely to require cytochrome c release from mitochondria, because it is controlled by Bcl-2 family proteins that function at this step of the pathway; it is unlikely, however, that there is widespread loss of mitochondrial outer membrane integrity, because the cells mostly remain viable. The ability of such cells to survive a low-level activation of caspase-3/7 also indicates that there are mechanisms to prevent conversion to full apoptosis, possibly through suppression of auto-amplification mechanisms that otherwise produce full caspase-3/7 activation, such as the inhibitory phosphorylation of caspase-9 [2] and caspase-2 [32] during mitosis. When mitotically delayed cells exit mitosis, a higher threshold for full apoptosis is likely to be restored as Bcl-2 and Bcl-xL are dephosphorylated and Mcl-1 levels recover through new synthesis. We propose that recovery mechanisms also reduce caspase-3/7 activity to non-stressed levels during interphase, perhaps through the activity of inhibitory proteins (IAPs) and/or the proteolytic turnover of the activated caspases. The majority of mitotically stressed cells are likely to survive with most of their constituent mitochondria intact, as has been observed in response to other apoptotic stimuli [33].
Subapoptotic caspase activity might induce a DDR in mitotically arrested cells through the generation of DNA strand breaks in a restricted manner by the apoptotic endonuclease CAD after cleavage of its inhibitor ICAD [17,34]. Interestingly, recent work has shown that a DDR is initiated at telomeres during a prolonged mitotic arrest [15], and telomeres might be particularly sensitive to CAD-dependent DNA strand breaks; alternatively, caspase-dependent cleavage of other proteins might lead to telomere deprotection. The repair of DNA double-stranded breaks is inhibited during mitosis, which prevents telomere fusions [35] and may allow CAD-generated breaks to accumulate during mitotic arrest. Subsequent signalling is likely to involve the recruitment of secondary factors to sites marked by γH2AX when cells exit mitotic arrest [35,36].
In addition to the DDR induced at specific foci during mitosis, massive DNA damage induced on individual lagging chromosomes during release from mitosis (an effect that, by contrast to foci formation, does not appear to be dependent on the period of prior mitotic arrest [37]) is likely to contribute to the effect of mitotic disruption in certain individual cells [37,38]. Furthermore, failure to complete nuclear envelope assembly in telophase is coupled to a widespread DDR in micronuclei [39]. Nevertheless, caspase-dependent DNA damage, which is restrained by Mcl-1 together with Bcl-2/Bcl-xL during normal mitosis, appears to be a major mechanism by which a prolonged mitotic delay affects subsequent cell cycle progression and cell proliferation.
The role of p53 in cellular responses to microtubule poisons has previously been unclear, but from our results it is now apparent that phosphorylation of the protein on Ser15, which is associated with the activation of its transcriptional activity [40], is induced by these drugs because of mitotic stress. We have found that, although Ser15 phosphorylation and the p53-dependent induction of p21 in response to microtubule poisons are blocked by inhibitors of the PIKKs, the induction of p53 protein is not, indicating a distinct mechanism for its transcriptional induction and/or stabilization of the p53 protein as a consequence of mitotic arrest. Downstream of p53, it is very likely that, in addition to p21, effectors involved in the control of cell proliferation or survival are induced transcriptionally in response to mitotic stress. Loss of p53 function during tumourigenesis might permit continued cancer cell proliferation by removing the G1 checkpoint following mitotic stress. Although we find that the increase in proliferation rate following depletion of p53 in U2OS cells (which are a useful model for p53 responses) is counteracted by an increase in apoptosis, in other cell types, this balance might differ owing to additional genetic changes, such that loss of p53 function could result in increased cell survival after the disruption of mitosis and might alter sensitivity to anti-mitotic drugs.
We propose that the caspase-dependent mitotic DNA damage pathway plays a role in the maintenance of normal chromosome fidelity and suppression of aneuploidy by inhibiting the proliferation and survival of aberrant cells. Conversely, suppression of this mitotic stress response in cancer cells is likely to sustain them by permitting continued proliferation despite delayed mitoses owing to polyploidy, aneuploidy or supernumerary centrosomes [41]. Inhibition of the mitotic stress response by Mcl-1 and Bcl-2/Bcl-xL suggests that these proteins can promote oncogenesis when they are over-expressed or stabilized not only through the prevention of apoptosis, but also through the suppression of both mitotic DNA damage signalling and subsequent activation of p53. Ordinarily, this response is likely to induce cell senescence as a consequence of a sustained G1 arrest, although not in cancer cells that are resistant to senescence, for instance, owing to loss of p14ARF(as in the U2OS cells used in our experiments [42]). Thus, inhibition of the mitotic stress response and the suppression of apoptosis might together permit the proliferation of cells with the increased rates of chromosome abnormalities commonly found in cancer.
Our results have potential implications for anti-cancer therapy. A very prolonged arrest in mitosis induced by a high concentration of a microtubule poison or ablation of Cdc20 can lead directly to cell death in some cell types [6,8,9], but such a prolonged arrest is unlikely to be sustained in patients undergoing chemotherapy, in which case the response of cancer cells to suboptimal doses of the drug and a delay (as opposed to an arrest) in mitosis are likely to be important for clinical effectiveness. We show that the mitotic stress response can be hyperactivated by a BH3 mimetic, which promotes the DDR, delays subsequent cell cycle progression and enhances apoptosis, thereby inhibiting cancer cell proliferation. The combination of a BH3 mimetic with taxol might therefore achieve an improved therapeutic index against proliferating cancer cells, particularly those in which the response to mitotic stress is suppressed by overexpression of Mcl-1 or another inhibitor of caspase activation.
We also find that inhibition of DDR kinases after mitotic stress has a similar overall effect to the BH3 mimetic, suggesting that the kinase inhibitors act by preventing recovery mechanisms resulting in a prolonged or enhanced response. This is evidenced by the strong DNA damage signals that accumulate after release from mitotic arrest when individual PIKKs are inhibited. Remarkably, there seems to be a commonality of cellular responses between apparently non-genotoxic anti-cancer drugs that target microtubules and genotoxic drugs or radiation that directly damage DNA because the former also induce a DDR through mitotic stress. Thus, drugs that target components of the DDR or DNA repair could enhance the effectiveness of anti-mitotic drugs.
Funding statement
This work was supported by a project grant (no. 12-0105) from Worldwide Cancer Research (formerly Association for International Cancer Research) and a programme grant (C275/A13424) from Cancer Research UK.
Dr Shubhra Rastogi provided U2OS cells synchronized in G1 by elutriation and commented on the manuscript.
- Received August 23, 2014.
- Accepted February 18, 2015.
© 2015 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited.


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"Like other employers, we used to be very concerned due to the increased employee lawsuits in California. In January 2004, after working with Robert Rediger on the first legal issue, we realized that our decision to retain Rediger McHugh was right. The attorneys provided us with legal services over the years, defending us on discrimination lawsuits, EEOC charges and DFEH complaints, etc. We also seek their general advice on preventative personnel matters and make personnel decisions with utmost confidence. We are very pleased with the knowledge, performance, support, professionalism and determination of Mr. Rediger, who answers all our questions and concerns with compassion, and for the peace of mind we have by having RM on our side."
"We have been working with Robert Rediger and his team for over 20 years. We routinely seek the input of attorneys at Rediger McHugh on day-to-day matters and they have guided us well in the preparation of employee handbooks and the development of sound, reasonable personnel policies. We comfortably rely on them for complicated issues as well, and they have provided expert and successful guidance in dealing with ADA claims and NLRB trials. I wouldn't trust my business interests to any other firm."
"Let me start by saying that Bob Rediger is the first person Carrie and I think about when I have a situation with an employee. All the advice and suggestions and defense that he has given Foothill Fire Protection has been spot on, and has saved our company time and money. I have had Mr. Rediger speak at our contractors' group a number of times, and have always learned something new in regard to employer-employee relationships. Thank you Rediger McHugh for always answering your phone."
"Having just completed a union negotiation, I can say with extreme confidence that Robert Rediger is a master of staying on point and delivering the essentials. He knows the law, has great attention to detail and delivered the most important elements. We ended up with a fair and balanced collective bargaining agreement that allows us the freedom and flexibility to continue to manage and grow our business. Rediger McHugh was outstanding!"
"I have worked with Robert Rediger with two different organizations, one was a union environment where he successfully guided us through negotiation of the contract as well as helped us to defend ourselves against frivolous grievances. In the second he was instrumental in heading off a potential age discrimination suit by a disgruntled sales person. He is always available to answer any question, we do not do anything in regards to employee relations without his guidance. I cannot more highly recommend Mr. Rediger and his firm for all of your employment law needs."
"I recently had the pleasure to use the services of Laura McHugh to assist a client of mine who needed expert help with a immediate labor issue. Laura worked tirelessly to resolve the matter, and was available to me and my client after hours to complete the difficult negotiations to the great advantage of our mutual client."


text: me and my ex united about a month ago. I really like her and want to kiss her chicks(we're not that much intimated to kiss each on lips,though we like each other) but don't know on which time and situation and will it be OK if I kiss her chicks or she will get angry on me? give me any suggestion guys..


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Rihanna shows off 'engagement ring' at Grammys
Rihanna, who was cuddling up to rapper Chris Brown at Grammy Awards, was pictured wearing an "engagement ring" while performing at the event.
The pair, who arrived separately on the red carpet, but were seated together couldn't hide their feelings for each other during the 55th annual Grammy Awards, the Mirror reported.
Photos: 55th Annual Grammy Awards
The Bajan singer cosied up to Brown and rested her head on his shoulder and happily posed for photos. But when she went on stage to perform, she was pictured with a ring on her engagement finger.
In a recent interview with Rolling Stone, Rihanna said that dating Brown again makes her happy and "if it's a mistake, it's my mistake."


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Search Engine Optimization & Marketing Statistics
Proof of the Power of Search Engine Marketing to Grow Your Business Now
Below are some statistics that establish the power and profitability of this marketing method. Like all marketing, it is not perfect or fool-proof, but it is the most effective way to grow your business. These stats also confirm that this area is becoming, if it isn't already, a standard part of strategy for companies across all industries.
- – Searchers will refine their search queries/terms, go back to search results after clicking on a listing or abandon the search completely 50% of the time [Microsoft Bing study]
- – 41% of searchers will change their search queries or go to another search engine completeley if they don't find what they want on the 1st page of results. 88% do so after 3 pages of unsuccesful search results. [Microsoft Bing study]
- – Search studies have found that women are more patient with search results, taking the time to read more of them and spending more time on sites before making a purchase decision.
- – One eye-tracking study found that searchers who have different objectives (shopping versus getting information) actually use search results differently. The 'Shopping/Commercial Intent' searchers clicked paid search listings as often as organic listings. The 'Info Gathering' searchers stuck to the organic results. [Enquiro study]
- – 62% of people click a result on the 1st page of search results. 90% click on a listing within the first 3 pages.
- – Despite Google's dominance, 2/3 of searchers visit at least 2 different search engines [Nielsen]
- – Search Engines bring prospects that convert (ie buy) at twice the rate of other online marketing methods. (WebSideStory Inc study)
- – Companies who used SEO on their site saw an average gain of 73% in traffic within 6 months. (MarketingSherpa study)
- – Companies who did the optimization in-house made a traffic gain of up to 38% in that same time period. (MarketingSherpa study)
- – Companies who hired an SEO firm to do the job saw a gain in traffic of up to 110%. (MarketingSherpa study)
- – Search Engine use is now the 2nd most popular online activity, led only by email. Email is used by 91% of the Internet, while search engines are used by 90%.
- – Search engines are used by 92% of web consumers to shop and/or buy.
- – 55% of all online purchases originate from Search Engines as opposed to only 9% from banners (NPD Group)
- – Search engine optimisation was the top method cited by Web site marketers to drive traffic to their sites (66%), followed by email marketing (54%) (Direct Marketing Association study)
Article written by Jonathan Cook


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- In the case of Chauke & Others v Lee Service Centre CC t/a Leeson Motors (1999), the employer dismissed 20 employees for misconduct in a situation where management was unable to pinpoint the actual perpetrator.
- The employer operated a panel beating shop where it repaired and spray painted accident damaged vehicles. Some of these vehicles became the target of malicious acts of sabotage. Although management could prove that the damage was committed by one or more of the employees, they were unable to identify which employee(s) where guilty.Management asked the staff to assist in identifying which employee(s) were guilty.
- When the staff failed to co-operate they were dismissed.
- The Labour Appeal Court agreed with the employer's decision.
Employment Practices Liability
This policy covers an employer's legal expenses and the awards made against it by the CCMA, Bargaining Councils and Labour Court for unfair labour practices such as unfair dismissal, sexual harassment, discrimination and failure to employ.
Question: Example 1 – Illustrate Derivative Misconduct?


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Hey, it's that season again. and really, I find myself not wanting anything this season. Except the opportunity to wish you all these things…
Comfort on difficult days
Smiles when sadness intrudes
Rainbows to follow the clouds
Laughter to kiss your lips
Sunsets to warm your heart
Hugs when spirits sag
Beauty for your eyes to see
Friendships to brighten your being
Faith so that you can believe
Confidence for when you doubt
Courage to know yourself
Patience to accept the truth
Love to complete your life
-from an author unknown-
Have a joyful season of wishing and giving everyone!!


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A former Richmond County Commission member has been sentenced to nearly three weeks in jail after shoplifting makeup and medicine from a pharmacy on Christmas Eve.
Paul William "Bill" Hiers, 69, was sentenced Monday, June 2, to 20 days in the Columbia County jail.
CVS employees at 3527 Old Petersburg Road witnessed Hiers putting CoverGirl makeup and a bottle of Aleve into his pants pockets before trying to leave the store, according to an incident report.
Hiers later told police he didn't want to stand in the long line so he decided not to pay. A woman with Hiers told police she handed him the items but was not aware he intended to steal the merchandise.
Hiers spent several hours in jail that day.
He served on the pre-consolidation Richmond County Commission from 1979-82 and was chairman in 1980.


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While we often hear about the negative impact social media has on children, the use of sites like Facebook, Twitter and Instagram is not a one-size-fits-all activity. Children use it in a wide variety of ways – some of which are adding value to their lives.Read More »
Tag Archives: cyber bullying
Education, social skills, and empathy to combat bullying
Bullying culture is widespread in Australian schools, yet policies are handed down to schools, only to be scrapped, causing confusion and impeding progress. What actually constitutes bullying? How do we fix it?Read More »
Cyber-bullying is pervasive, but that doesn't mean we should accept it
Bullying has long been a problem within schools, and it has been sad to watch bullying morph into a new phase as technology has advanced - Cyber-bullying.Read More »
Body image: taming the beast with media literacy
In 2008, a Victorian government document titled Because Mental Health Matters Consultation Paper argued for 'further development of positive body image programs, in conjunction with healthy eating programs' as a priority activity within the 'early childhood and school' setting.Read More »
Schools, sign up for eSafety Challenge program
The Office of the Children's eSafety Commissioner, has released ClassAct 50 Challenge, designed to help children take control of their online safety, prompt thinking around respectful relationships and to know where to go to find support if things go wrong.Read More »
Finland strikes again, this time at bullying
The Finnish education system is coveted by teachers, parents and informed students all over the world. After introducing reforms more than 40 years ago, Finland's education system has been characterised by a teaching profession that is harder to enter than medicine.Read More »


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Valentine's Day is just around the corner so I thought today would be a good time to roundup some of my favorite engravable "initials" jewelry and show you that there's more to Stella & Dot than just #5RingsToStopYelling. And if a link to this post, or any of the links within the post, happens to, somehow, get emailed to your husband, then it'll be a win-win-win. Your hubby will be guaranteed a victory with his V-day gift this year and you will get a gorgeous piece of bling … [Read more...]
Positive Parenting: A #5RingsToStopYelling Update
Ok guys, it's been about a week now since my 5 rings arrived (check out these slim gold, silver, and rose gold rings if you'd like to build a set of your own) - time for an update. (Before getting to that, if you'd like some background on this challenge go here for why we're doing this.) First, and probably most importantly, I'm still at it! One of the scariest things about committing (quite publicly) to doing something outside of your comfort zone is wondering if you can actually follow … [Read more...]
How 5 Rings Are Going to Make Me a Better Mom this Year
How many of you resolved to yell less at your kids this year? Actually, let me amend that, to just be nicer to your kids? I ask because I've seen a whole lot of stuff going around the web lately about how to stop yelling. Apparently yelling is the new spanking for our generation. I get it. Yelling at your kids totally sucks. Even while you're doing it you are already regretting it, yet you can't seem to make it stop. Nobody wants to parent that way - for sure. But I'd bet that most of … [Read more...]


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Wedding Officiant in and around Alcalde, Nm.
Life's Moments are those times in life that we decide to make a special recognition of life altering events. Weddings are celebrations, weddings are ceremonies, yet most of all weddings are a new beginning, a minute in time to be valued. Having the best Minister is very important.
For these minutes, we offer comprehensive Wedding Minister solutions, Getting Married In officiating, and wedding celebration services. From the very standard to modern services, our wedding celebrations and officiating solutions are designed to show your wants, wishes and dreams. We will help you to craft an event, spiritual or secular that will be a treasured memory for many years to come.
Pastoral solutions as well as wedding celebration officiating in the Alcalde, Nm location is our specialty, but we could travel if the requirement arises. We give a full range of Getting Married In packages; from elopement on the courthouse steps to the most sophisticated formal wedding you might want.
We additionally provide the finest in Justice of the Peace or secular weddings. These civil ceremonies provide their very own type of celebration, whether it be with family members or simply a couple and also a couple of witnesses, your wedding is an unique moment to be valued.


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|1,246 sq. ft.
|Property type
|Minimum Stay
|3 - 7 nights
Mala Pua ~ Kauai Vacation Condo, Vacation Rental Near Famous Poipu Beach.
* Easily accessible ground floor condo with Central Air Conditioning
* Steps away from the resort pool - BBQ at poolside
* 5 minute walk to Poipu Beach ~ great for adults and small children alike
* Beach gear in the condo for your use
* Short walk to the Grand Hyatt
* Base rate is for 4 people, maximum 5. Please view "RATE NOTES"
Mala Pua Regency ("garden" in Hawaiian) is nestled in abundant tropical greenery with two lanais to relax and enjoy the island pace. This easily-accessible ground floor condo has all the comforts of home including central air conditioning, plus it is just steps to the pool and a short walk to wonderful beaches.
With generous natural light and verdant foliage, the living room offers two sofas, a wicker desk and entertainment center.
Dine comfortably at the fanciful "pineapple" pedestal table; plus casual seating at the breakfast bar.
Central A/C throughout the home plus ceiling fans in each room for maximum comfort.
The deep rich granite counters make an inviting stage for whipping up culinary creations in the fully-equipped kitchen.
The master suite has a king bed, TV and private lanai. The luxurious marble bath with twin sinks boasts a large walk-in shower and deep jetted tub.
The guest bedroom has 2 twin beds that can be converted into a king-sized bed. A full bath with tub/shower combo is across the hall.
Premium channel TV service, high speed wireless internet, DVD/CD players and private phone are provided.
Beach towels, mats, beach chairs, cooler and assorted gear are available for your use.
Complimentary Play N' Pack for babies provided upon request.
The prestigious Poipu-Kai resort situated on the South Shore of Kauai, features 70 acres of tropical gardens. It is just a short walk through the resort grounds to Shipwreck beach or Poipu Beach Park (a snorkeler's paradise). The resort has nine tennis courts. There is a shop for all your tennis needs, including rentals. After the game relax in one of the Jacuzzi spas or swimming pools. You can barbecue your "catch of the day" in one of the well maintained grills near the pool.
Kauai Beaches: Poipu Kai resort fronts on two well known beaches... Shipwreck Beach and Brennecke Beach, each an excellent bodysurfing beach. Next to Brennecke beach is Poipu Beach Park, rated the best beach in North America for four years running. It features a sheltered "baby beach" for toddlers and parents, a fully equipped children's playground, and a great snorkeling and swimming beach with every variety of tropical fish usually only seen in aquariums. Surfing is also an excellent option at Poipu Beach with a surf shop and board rentals just across the street from the beach.
Kauai Restaurants: Brennecke's restaurant is a great place for lunch or dinner at the beach; good burgers, fish and drinks. They have a great crab meal for dinner. Don't forget Lapperts' ice cream for a real treat. It's fresh made in Hanapepe and definitely a landmark destination. They are located throughout the island: Hanapepe, Poipu, Hanapepe, Kalaheo, and Kapa'a, etc.
Island activities: For the shoppers there are some very nice boutiques in the Poipu Village shopping center just West on Poipu Road. There are also boutique shops at the surrounding resorts, e.g., Hyatt, Sheraton and the Marriott. Food shopping is nearby at the Big Save supermarket in Koloa, just a couple of miles up the bypass road. If you are energetic and want some exercise Kauai offers a great series of hikes and drives, some with breathtaking views. Try the hike along the beach and cliffs east past the Hyatt. It is about 4 miles and the reward is a wonderful white sand beach. There is a spectacular hike/drive up the Waimea Canyon (the grand canyon of the Pacific). At the end of the road hike the trail about 4 miles which will take you to a lookout over the Na Pali Coast. Also refer to the Kauai Guide Book located in the house for more fun things to do.
This VRBO partner has demonstrated and is committed to fast response times, the best rates, and a great traveler experience.
Juliana and Jim own 2 homes in Kauai and manage a number of other beautiful homes and condos. They began visiting the Garden Island in 1997 and now are permanent residents. Juliana is a licensed real estate broker in Hawaii. Her special enjoyment is gardening. She loves to share the many wonderful features of Kauai with her visitors, especially the great beauty of the local botanical gardens. Jim is a builder of many years and now enjoys semi-retirement while keeping his hands busy with the occasional remodel.
This was our favorite of the three condos we rented in Hawaii. Two full bedrooms and two full baths with the master having a spa tub and walk in shower. It had central air conditioning which is difficult to find in the islands. The washer dryer was full sized . Sunken living room. Kitchen very well equipped and both bar stools and a table for eating. Pool view bottom floor with a lanai off the living room and the master bath. The owners were the easiest to communicate with because they clearly let you know their direct cell phone number and you don't have to communicate through Home Away which is a total pain. Poipu is a great place to stay because it is the sunniest. Nice beach a short distance away. Highly recommend the taco shack called DA CRAC and the luau at the Grand Hyatt. Wonderful stay with wonderful and friendly reps for the condo.
We had the best family vacation we have ever experienced, and I'm not just saying that as an expression.
First, location: the pool is right outside the door, and not used much at all, we were almost always alone, and our kids loved the access to the pool. The situation of the unit is the best in the whole resort in my opinion, secluded and surrounded by tropical plants and foliage, and quiet, no road or traffic noise. The lanai off the master suite is amazing, feels like you are in the middle of the jungle. You can walk to several great meals, Brenneckes (highly recommended this was our go to place), Tidepools, Kalapaki Joes, Brenneckes deli (great sandwiches and breakfast) and just a very short drive to the shopping center with Puka dogs and sushi, or a bit further to the Beachhouse for a sunset dinner (this is a must!) walking the green belt pathway to several of the beaches in the immediate area is lit at night, has a variety of wildlife, frogs (don't touch them they excrete toxins!) kids enjoyed lizard hunting, without much luck, all kinds of birds and beautiful landscaping and flora. We loaded up the provided beach gear, which was awesome, chairs, umbrella, mats, sand toys, cooler, and took the short 5 minute drive down to Poipu Beach many days, it's an awesome beach! Snorkeling, you can see tons of fish in the very protected section of water. Sometime the kids and wife would walk back, and I would haul the gear. Our very first day we walked to the beach before dinner and got to see sea turtles sleeping on the beach! It was an amazing experience.
The condo: everything we needed was provided. The ac was a god sent, even set at 75 it felt amazing coming home after a day at the beach and 85 deg temps. The slat blinds were great. Everything you could need in the kitchen, ice maker (enough capacity to fill the beach cooler) and blender, key pieces of equipment. The unit was very clean upon arrival which isn't always the case with these bigger condos. The painless self check in/out and keypad door lock were great as well. The unit is beautifully and thoughtfully decorated. The unit managers were great, even after leaving there was a slight issue with some damage to a cabinet that we were not aware of, and it was handled wonderfully by them.
I could go on and on but am limited by characters! Great unit, we will return next year unless we can drag more family members along with us, we will look for a bigger unit in the same resort next year!
Great location, between Hyatt and Poipu beach. Pool and jacuzzi just steps away. Convenient reserved parking stall. Well kept interior. Air conditioned! Would definitely recommend and stay here again. Management very accommodating. Only criticism would be that the master bed has probably reached the end of its lifetime.
This condo is perfectly located close to the pool and hot tub and has great privacy. There is a very nice lanai off the living with a patio table which was perfect for eating breakfast and dinner and enjoying the sunsets. There is another lanai off the master suite which is also very private. The master bath is beautiful with a huge shower and a jetted tub. The kitchen had everything that we needed. The design of this condo is perfect with a very open kitchen/dining/living concept and it is very well furnished.
We had a great stay at this condo. It was much better furnished than we had expected and so thoughtfully equipped that we felt we were at home. We made use of the cooler to take a picnic to the beach, and our kids were grateful for the boards left there for our use. In the evenings, we hung out at the pool and slept soundly as it was very quiet. One never knows what one is renting but the reviews helped us pick this condo, and we are so grateful to have found it. We had the best stay, and it was far more spacious than a hotel. Our kids want to come back to this condo next time.
This condo was wonderful. The unit is upscale and very nicely furnished. The complex is quiet, beautifully landscaped, and peaceful. There is a pool just steps from the condo for convenience, yet the condo is quiet inside. The condo is perfect for two couples or a couple with two children. The kitchen is roomy enough for two to prepare meals and clean up. We would stay here again.
The prestigious Poipu-Kai resort situated on the South Shore of Kauai, features 70 acres of tropical gardens. It is just a short walk through the resort grounds to Shipwreck beach or Poipu Beach Park (a snorkelers paradise). The resort has nine tennis courts. There is a shop for all your tennis needs, including rentals. After the game relax in one of the Jacuzzi spas or swimming pools. You can barbecue your "catch of the day" in one of the well maintained grills near the pool.
Beaches: Poipu Kai Resort fronts on two well known beaches: Shipwreck Beach and Brennecke Beach, each an excellent bodysurfing beach. Next to Brennecke beach is Poipu Beach Park, rated the best beach in North America for four years running. It features a sheltered "baby beach" for toddlers and parents, a fully equipped children's playground, and a great snorkeling and swimming beach with a multitude of tropical fish.
Other Activities: Great zip line tours!