Cell Cycle Assays-Part I

From alleleblog: http://allelebiotech.com/blogs/

This is the first part of a series of blogs about using fluorescent proteins in cell based assays with established examples, a common theme here at the AlleleBlog.

FUCCI Cell Cycle Sensor

The FUCCI Cell Cycle Sensor is composed of a red (RFP) and a green (GFP) fluorescent protein fused to different regulators of the cell cycle: cdt1 and geminin.

During the cell cycle, these two proteins are ubiquitinated at different time points by specific ubiquitin E3 ligases, which tag them for degradation in the proteasome. The E3 ligases’ activities are regulated temporally and result in the biphasic cycling of GERMINI and CDT1 levels during the cell cycle. In the G1 phase of the cell cycle, GERMINI is degraded; therefore, only CDT1 tagged with RFP is present and appears as red fluorescence within the nuclei. In the S, G2, and M phases, CDT1 is degraded; only GERMINI tagged with GFP is present, resulting in cells with green fluorescent nuclei.

During the G1/S transition, when CDT1 levels are decreasing and GERMINI levels increasing, both proteins are present, so are the tagged fluorescent proteins. When the green and red images are overlaid, nuclei fluoresce yellow. This dynamic color change, from red-to-yellow-to-green, represents the entire cell cycle. This representation can be used to study the effects of elements that may influence cell cycles.

Sakaue-Sawano A, Kurokawa H, Morimura T, Hanyu A, Hama H, Osawa H, Kashiwagi S, Fukami K, Miyata T, Miyoshi H, Imamura T, Ogawa M, Masai H, Miyawaki A.Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell. 2008 Feb 8;132(3):487-98.

CCNB1-CyclinB(NT)-GFP

In late S phage, CCNB1 promoter will be switched on to drive the expression of Cyclin B N-terminus-GFP expression; thereafter the fluorescent signal will be switched off at the destruction box in Cyclin B N-terminus at the end of Mitosis phase. During the intervening phase the fusion reporter protein will translocate from cytoplasm to nucleus by the cytoplasmic retention signal in the Cyclin B N-terminus.

Thomas N. Lighting the circle of life: fluorescent sensors for covert surveillance of the cell cycle. Cell Cycle. 2003 Nov-Dec;2(6):545-9.

GFP-PCNA/YFP-PCNA
GFP-PCNA, a fusion of GFP and PCNA, has been widely used as a convenient tool to monitor the progress of S phase. At the onset of S phase, GFP-PCNA translocates into the nucleus; at mitosis the nuclear envelope breaks down and the nuclear accumulation of PCNA-GFP dissipates.

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The chairman of the Norwegian Nobel Committee: Why We Gave Liu Xiaobo a Nobel

New York Times, Oct. 22, 2010

我们为什么颁奖给刘晓波

(Thorbjorn Jagland is the chairman of the Norwegian Nobel Committee)

在中国当局对诺 贝尔委员会选择刘晓波(被监禁的政治活动家)作为2010年和平奖得主的谴责无意中说明了为什么值得捍卫人权。

当局声称，任何人都无权干 涉中国的内部事务。但他们错了：国际人权法和标准高于民族国家，而且国际社会有责任确保它们得到尊重。

现代国家制度是国家主权观念演变而 来的，其又是由1648年的威斯特伐利亚和平协议建立的。当时，主权被认为是在一个专制统治者中体现。

但有关主权的想法已经随时间改变 了。美国独立宣言和法国的人权和公民权宣言取代了独裁者控制下的人民的主权作为国家权力以及合法性的来源。

在上个世纪，主权的概念再次改 变了，随着世界从民族主义转移到国际主义。在两个灾难性的世界大战后成立的联合国，让会员国承诺通过和平手段解决争端，并在世界人权宣言中确定全体人民的 基本权利。宣言中说，民族国家将不再有最终的、无限的权力。

今天，普遍人权对世界各地的任意多数提供了一种限制，无论是民主与否。在议会 中的一个多数并不能决定伤害一个少数群体的权利，也不能投票给损害人权的法律。即使中国不是一个宪政民主政体，它是联合国的会员国，而且它已经修改了宪法 以符合世界人权宣言。

但是，刘先生的监禁是清楚地证明，中国的刑法是不符合其宪法的。他被判定犯有“散布谣言，诽谤或者其他手段，颠覆国 家政权，推翻社会主义制度。”但在普遍人权为基础的国际社会，杜绝意见和谣言不是一个政府的工作。各国政府有义务确保自由表达意见的权利——即使说话者主 张不同的社会制度。

这些权利都是诺贝尔委员会捍卫已久的，通过授予那些挣扎着保护它们的人以和平奖，包括安德烈萨哈罗夫为他坚持反对苏联 的人权侵犯，和马丁路德金牧师博士为他争取在美国的公民权利。

毫不奇怪，中国政府已经严厉批评该奖，声称诺贝尔委员会非法干涉其内部事务 和在国际公众的眼睛中羞辱了它。相反，中国应该感到自豪，它已变得强大到足以成为辩论和批评的主体。

有趣的是，中国政府并不是唯一一个批 评诺贝尔委员会的。有些人说，颁奖给刘先生实际上可能恶化中国人权倡导者的境况。

但是，这种说法是不合逻辑的：它导致的结论是我们最好通 过保持沉默来促进人权。如果我们对于中国保持沉默，谁将会是下一个国家要求它保持沉默和不干涉的权利？这种做法将把我们放在一个走向破坏世界人权宣言和人 权的基本原则的道路上。我们绝不能保持沉默。任何国家都没有权无视其国际义务。

中国有充分的理由为它在过去20年来的成就感到自豪。我们 希望看到这一进步继续下去，这就是为什么我们颁发和平奖给刘先生。如果中国是要推进与其他国家的和谐，成为维护国际社会的价值观的一个重要伙伴，它必须首 先给予其所有公民言论的自由。

一个人仅仅因为他表达了他的意见而正在被监禁11年，这是一个悲剧。如果我们要走向阿尔弗雷德诺贝尔所说的 国家的博爱，那么普遍人权必须成为我们的试金石。

Why We Gave Liu Xiaobo a Nobel
By THORBJORN JAGLAND
Published: October 22, 2010

THE Chinese authorities’ condemnation of the Nobel committee’s selection of Liu Xiaobo, the jailed political activist, as the winner of the 2010 Peace Prize inadvertently illustrates why human rights are worth defending.

The authorities assert that no one has the right to interfere in China’s internal affairs. But they are wrong: international human rights law and standards are above the nation-state, and the world community has a duty to ensure they are respected.

The modern state system evolved from the idea of national sovereignty established by the Peace of Westphalia in 1648. At the time, sovereignty was assumed to be embodied in an autocratic ruler.

But ideas about sovereignty have changed over time. The American Declaration of Independence and the French Declaration of the Rights of Man and of the Citizen replaced the control of the autocrat with the sovereignty of the people as the source of national power and legitimacy.

The idea of sovereignty changed again during the last century, as the world moved from nationalism to internationalism. The United Nations, founded in the wake of two disastrous world wars, committed member states to resolve disputes by peaceful means and defined the fundamental rights of all people in the Universal Declaration of Human Rights. The nation-state, the declaration said, would no longer have ultimate, unlimited power.

Today, universal human rights provide a check on arbitrary majorities around the world, whether they are democracies or not. A majority in a parliament cannot decide to harm the rights of a minority, nor vote for laws that undermine human rights. And even though China is not a constitutional democracy, it is a member of the United Nations, and it has amended its Constitution to comply with the Declaration of Human Rights.

However, Mr. Liu’s imprisonment is clear proof that China’s criminal law is not in line with its Constitution. He was convicted of “spreading rumors or slander or any other means to subvert the state power or overthrow the socialist system.” But in a world community based on universal human rights, it is not a government’s task to stamp out opinions and rumors. Governments are obliged to ensure the right to free expression — even if the speaker advocates a different social system.

These are rights that the Nobel committee has long upheld by honoring those who struggle to protect them with the Peace Prize, including Andrei Sakharov for his struggle against human rights abuses in the Soviet Union, and the Rev. Dr. Martin Luther King Jr. for his fight for civil rights in the United States.

Not surprisingly, the Chinese government has harshly criticized the award, claiming that the Nobel committee unlawfully interfered with its internal affairs and humiliated it in the eyes of the international public. On the contrary, China should be proud that it has become powerful enough to be the subject of debate and criticism.

Interestingly, the Chinese government is not the only one to criticize the Nobel committee. Some people have said that giving the prize to Mr. Liu may actually worsen conditions for human-rights advocates in China.

But this argument is illogical: it leads to the conclusion that we best promote human rights by keeping quiet. If we keep quiet about China, who will be the next country to claim its right to silence and non-interference? This approach would put us on a path toward undermining the Universal Declaration and the basic tenets of human rights. We must not and cannot keep quiet. No country has a right to ignore its international obligations.

China has every reason to be proud of what it has achieved in the last 20 years. We want to see that progress continue, and that is why we awarded the Peace Prize to Mr. Liu. If China is to advance in harmony with other countries and become a key partner in upholding the values of the world community, it must first grant freedom of expression to all its citizens.

It is a tragedy that a man is being imprisoned for 11 years merely because he expressed his opinion. If we are to move toward the fraternity of nations of which Alfred Nobel spoke, then universal human rights must be our touchstone.

Thorbjorn Jagland is the chairman of the Norwegian Nobel Committee.

DNA Repair Pathway Factors in Cell-Based Screening for Restoring Patients’ Sensitivity to Cancer Therapies

Cancers undergoing therapies may develop resistance to treatment. Many current cancer treatments, such as cisplatin, function by creating DNA damage, particularly to fast-dividing cells, i.e., most cancer cells. These treatments may be rendered ineffective by DNA-damage response pathways. Cancer resistance to therapies may come from increased activity in nonhomologous end joining, decreased functions of mismatch repair, or reactivation of the Fanconi anemia (FA)/BRCA DNA-damage response pathway, etc. Ironically the loss of function of some of these DNA-damage repair factors may have partially caused the cancer formation in the first place. Regaining their functions in cancer cells possibly contribute to drug resistance. Molecules that disrupt FA/BRCA pathway or other DNA-damage responses could be used to help restore therapy sensitivity.

Like many proteins that function in DNA-damage repair complexes, FANCD2, a member of the FA pathway factor group, is targeted towards chromatin following damage to DNA in a process called foci formation. There have been recent studies that monitored the foci formation of GFP-FANCD2 in small molecule library screening and identified inhibitors to FANCD2 as candidates for a cancer therapy sensitizer. The assays can be improved in a number of ways. There are fluorescent proteins (FPs) that are much brighter than EGFP for increased sensitivity. For instance, the monomeric green FP mWasabi is about 2-3 fold brighter than EGFP, with narrower emission peak, and is more stable under acidic environment. The newly developed lancelet YFP (LanYFP, developed/introduced by Allele Biotech) is astonishingly 10 times brighter than EGFP. Since it has a longer excitation and emission wavelength, it should inherently have a better signal to noise/background ratio compared to EGFP because cells autofluoresce less in long wavelengths. The improved brightness would also help in this respect. The fold difference between foci and LanYFP background will be the same as EGFP, but the contrast will still probably be better because of less autofluorescent background and significantly higher fluorescence reading in foci.

Other factors that may be used as a screening target when fused to effective FPs may probably include:

1) Homologous recombination (HR)
a. End Resection
MRN complex (MRE11, RAD50, NBS1)
CtIP, RPA, ATM, ATR, Exo1, BLM, RMI1, TopIIIa, DNA2, BRCA1
b. Synapsis
RAD51, BRCA2, PALB2, RAD51B, RAD51C, RAD51D, RAD51AP1, XRCC2, XRCC3, RAD54, RAD54B
c. DNA synthesis
DNA polymerase delta, PCNA
2) Nonhomologous End Joining (NHEJ)
Ku70/Ku80, DNA-PK, Ligase IV, XRCC4, XLF
3) Fanconi Anemia Pathway
FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, FAAP100, FANCM, MHF, FAAP24, FANCD2, FANCI, FAN1, FANCN, FANCJ, FANCM

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Original post: http://allelebiotech.com/blogs/2010/10/dna-repair-pathway-factors-in-cell-based-screening-for-restoring-patients%E2%80%99-sensitivity-to-cancer-therapies/

Dr. Liu Xiaobo Wins 2010 Nobel Peace Prize

For "his long and non-violent struggle for fundamental human rights in China".

"From my personal angle, I feel in a dictatory society if you want to be a person with dignity, if you want to be a honest person, fight for human-rights improvement, fight for free speech, being ... [in prison] is part of what you are undertaking, and there is nothing to complain," Liu Xiaobo told CNN in 2007, while he was between a series of house arrests."

"Since you chose to do this, you must have a preparation for being in prison," he said. "Entering the prison you must face these things peacefully, not complain [about] others. I even don't complain [about those] ... who arrested me, because this is their inevitable action. I can also not let them arrest me if I chose other way." said Liu before the current prison sentence.  He served one previously for the 89 student movement.

He lost his freedom, willingly, twice, for the freedom of many others.  He is a living example of honor, dignity, bravery, and idealism.  Proud to have shared battle field with the hero 21 years ago.